Embodiments disclosed herein relate to methods for purifying proteins, including antibodies, such as IgG and IgM antibodies.
Purification of proteins typically begins with a clarification step in which cells and debris are removed so that the remaining supernatant can be processed by methods that would otherwise be hampered by their presence. Their removal commonly involves physical methods such as centrifugation and filtration. This step sometimes involves the use of filtration materials with anion exchange capabilities, or the addition of anion exchange particles or soluble polymers directly to the antibody-containing harvest (Gagnon, P., Purification Tools for Monoclonal Antibodies, Validated Biosystems, Tucson, 1996; Kuczewski, M,. et al, Biopharm Int. 23(3) (2010) 20-25; Kuczewski, M., et al, Biotechnol. J., 6 (2011) 56-65.
Secondary treatment of physically clarified cell culture harvests with allantoin, soluble organic cations, and mixed particles has been described (Gan, H. et al J. Chromatogr. A, 1291 (2013) 33-40). This approach particularly reduced the content of chromatin expelled by dead cells and the levels of aggregates associated with chromatin, but three chromatography steps were subsequently needed to achieve the desired purity. Allantoin is an FDA-approved anti-inflammatory agent used widely in over-the-counter healthcare products. It is known to remove endotoxin from protein solutions, including from solutions of IgG, apparently through hydrogen bonding (Vagenende et al, ACS. Appl. Mater. Interfaces, 22 (2013) 4472-4478; Vagenende et al, J. Chromatogr. A 1310 (2013) 15-20).
Partial purification of IgG antibodies by contaminant co-precipitation with caprylic acid (octanoic acid) has been disclosed (Chantuin, A., et al, Arch. Biochem. Biophys. 89 (1960) 218-220; McKinney, M. et al, J. Immunol. Met., 96 (1987) 271-278). The fatty acid binds to all proteins but tends particularly to precipitate acidic non-IgG contaminants (Gagnon supra; Morais, V., et al, Biotechnol. Appl. Biochem., 59 (2012) 50-54). The mechanism and process development guidelines for application to cell culture harvests have been indicated (Gagnon supra), including basic variables such caprylic acid concentration, pH, salt concentration, temperature, and the need for a subsequent chromatography step to remove residual caprylic acid from the soluble IgG preparation. The technique is most often described to prepare crude samples for subsequent purification by other means (Gagnon supra; Y. Yigsaw et al, 2008, Improving upstream feed stock to downstream operations, Recovery of Biologics Conference XIII, Quebec; Arunakumari, A. et al, US Patent Application 20120101262 A1), but has also been applied as a polishing step following antibody capture by protein A affinity chromatography (Y. Brodsky et al Biotechnol. Bioeng. 109 (2012) 2589-2598).